Polyacrylamide (PAM) is referred to as an “additive for different purposes” and has wide applications in many industrial fields, such as tertiary oil recovery, water treatment, paper making, mining, metallurgy, coal cleaning and manufacture of super water-absorbent resins and so on. The monomer of polyacrylamide is acrylamide (with a molecular formula of C3H5NO, and a structural formula of H2C═CHCONH2), which is generally produced through catalytic hydration using acrylonitrile as raw material. The hydration method for acrylamide experienced three stages of development, i.e. sulfuric acid catalysis, copper catalysis and microbiological process. Currently, the microbiological process has become the primary method of acrylamide production due to its advantages, such as normal temperature and normal pressure for the reaction, low energy consumption, simple operation, safety, high conversion rate of acrylonitrile, high concentration and purity of the product, etc.
The method for producing acrylamide using microbiological process mainly focuses on screening and domestication of bacterial strains, as well as the establishment and optimization of the manufacture techniques.
For the finding, domestication, mutagenesis and reconstruction of the production strains, Hideaki Yamada group in Japan discloses a wild-type Rhodococcus rhodochrous J-1 in the patent with the title “BIOLOGICAL PRODUCTION METHODS OF AMIDES ” (Chinese Patent No.: ZL88106735). Nippon Chemical Industrial Co., Ltd. discloses Rhodococcus sp. S-6, Arthrobacter oxydans and Microbacterium flavum in the patent with the title “PREPARATION OF AMIDES USING MICROORGANISMS” (Chinese Patent No.: ZL86100062). CIBA SPECIALTY CHEMICALS WATER TREATMENTS LIMITED discloses Rhodococcus rhodochrous NCIMB 41164 strain and its mutants in the patent with the title “STRAIN OF RHODOCOCCUS RHODOCHROUS NCIMB 41164 AND ITS USE AS PRODUCER OF NITRILE HYDRATASE” (Chinese Patent Application No.: 200480035487.1). Ashland Licensing and Intellectual Property LLC discloses Rhodococcus rhodochrous strain M33 in the patent with the title “METHOD FOR CULTURING THE NITRILE HYDRATASE-PRODUCING STRAIN RODOCOCCUS RHODOCHROUS M33” (Chinese Patent Application No.: 200480043243.8). Shanghai Pesticide Research Institute describes a wild-type Corynebacterium propinquum and its domesticated strain for acrylamide production by microbiological process in the patent with the title “PRODUCTION OF ACRYLAMIDE WITH MICROBIAL CATALYSIS” (Chinese Patent Application No.: 03115536.7). Tsinghua University discloses a method of obtaining a tolerant nitrile hydratase in the patent with the title “THE ORIENTATION CULTURE METHOD FOR IMPROVING THE PRODUCT-TOLERANCE OF NITRILE HYDRATASE” (Chinese Patent No.: ZL 03130658).
In recent years, in the gene research and engineering on the conversion of acrylonitrile to acrylamide, Japanese Mitsubishi Chemical Corporation filed a patent with the title “NOVEL PROTEIN WITH NITRILE HYDRATASE ACTIVITY AND THE GENE ENCODING THE SAME” for the nitrile hydratase gene and the protein from the genus Rhizobium (Chinese Patent Application No.: 93106122.9). Mitsui Chemicals Co., Ltd. filed patents with the titles “A PROTEIN INVOLVED IN ACTIVATION OF NITRILE HYDRATASE AND THE GENE ENCODING THE SAME” (Chinese Patent No.: ZL99106291.4) and “NOVEL NITRILE HYDRATASE” (Chinese Patent No.: 02156180.X), respectively, for a nitrile hydratase protein and its encoding gene from Pseudonocardia thermophila JCM3095, and also investigated the expression of the gene in the recombinant Escherichia coli. Degussa A G filed a patent with the title “NITRILE HYDRATASE OF RHODOCOCCUS” (Chinese Patent Application No.: 200580008206.8), and Mitsubishi Rayon Co., Ltd. filed a patent with the title “IMPROVED NITRILE HYDRATASE” (Chinese Patent Application No.: 200580016665.0), disclosing a method for improving the thermotolerance of nitrile hydratase by site-directed mutagenesis. Tsinghua University obtained a nitrile hydratase gene from Nocardia sp. and filed a patent with the title “A NITRILE HYDRATASE, ITS ENCODING GENE AND THE USE THEREOF” (Chinese Patent No.: ZL 200410042576.0), wherein the mutation and high expression of the gene in recombinant Escherichia coli were further studied.
In addition to nitrile hydratase, in the strains that produce acrylamide using nitrile hydratase, there is amidase which can further convert acrylamide produced by nitrile hydratase catalysis into by-product acrylic acid, thereby seriously affecting the quality and yield of acrylamide, as well as increasing the purification difficulty and the production costs. Nitrile hydratase and amidase genes are usually organized in a same large gene cluster (such as in Pseudomonas chlororaphis B23, Rhodococcus sp. N771, Rhodococcus rhodochrous J1, Rhodococcus sp. RHA1 and Bacillus sp. BR449) or in independent gene clusters (such as in Nocardia farcinica IFM 10152 and Rhodococcus rhodochrous sp. M8) in the chromosomal genome of the strains (Brandao et al., Applied and Environmental Microbiology, 2003, 69 (10): 5754-5766; Ryabchenko et al., Genetika, 2006, 42(8):1075-82.). If the amidase gene is inhibited or knocked-out, the by-product acrylic acid can be avoided to be produced in a large amount during the production of acrylamide, thereby significantly reducing the burden of isolation and purification in the refining unit and the production cost.
In the present invention, the amidase gene is knocked-out from a nitrile hydratase-producing bacterial strain, so as to suppress the generation of by-product acrylic acid during the production of acrylamide.